Human T-cell leukemia virus subgroup I (HTLV-I) is a retrovirus closely associated with adult T-cell leukemia/lymphoma (ATL) (Poiesz et al., 1980, Proc. Natl. Acad. Sci. U.S.A., 77, 7415-7419). It is also linked with neurological diseases designated as tropical spastic paraparesis and HTLV-I-associated myelopathy (HAM) (Gessain et al., 1985, Lancet ii, 407-410). Human T-cell leukemia virus subgroup II (HTLV-II) was first isolated from a patient with T-cell hairy cell leukemia (Kalyanaraman et al., 1982, Science, 218, 571-573). It is associated with a T-cell variant of hairy cell leukemia.
The HTLV-I and HTLV-II genomes exist as proviruses in the human chromosomal DNA of leukemia cells. Their complete nucleotide sequences have been elucidated (Seiki et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80, 3618-3622; Shimotohno et al., 1985, Ibid., 82, 3101-3105). Like other retroviruses, the HTLV-I and HTLV-II genomes are flanked by LTR (long-terminal-repeats) structures which are believed to play an essential role in the integration of the proviral DNA into the host chromosomal DNA. Four major genes have been identified and occupy the following relative positions in the genome: LTR-gag-pol-env-pX-LTR (Seiki et al.; Shaw et al., 1984, Proc. Natl. Acad. Sci., 81, 4544-4548). The gag gene codes for a 48,000-dalton-precursor protein (often referred to as p53) consisting of 429 amino acids (433 for HTLV-II). This precursor protein is cleaved into at least three smaller proteins (FIG. 2). The pol gene codes for the viral reverse transcriptase. The env gene presumably codes for a 54,000-dalton protein which is glycosylated and later cleaved into two glycoproteins named gp46 and gp21. (Slightly different molecular weights have been reported for what are most probably the same env gene protein products). The last coding region is called pX and is believed to code for four proteins (tax.sub.1, tax.sub.2, rex.sub.1, rex.sub.2) involved in gene expression and regulation.
As depicted in FIGS. 1 and 2, the env proteins and the gag proteins of HTLV-I and of HTLV-II share a high degree of amino acid sequence homology. Furthermore, serologic cross-reactivity has been reported between the proteins of HTLV-I and those of HTLV-II (Lee et al., 1984, Proc. Natl. Acad. Sci., 81, 7579-7583). Despite this structural resemblance and cross-reactivity, some regions of these env and gag proteins are recognized only by antibodies present in a patient infected by either the HTLV-I or the HTLV-II virus.
HTLV-I and HTLV-II both differ from the human immunodeficiency viruses (HIVs) in their morphologic and genetic structures. As a result, antibodies to the HIV proteins should not cross-react with HTLV-I and HTLV-II antigens. However, some serum samples taken from patients with AIDS have shown some degree of cross-reactivity with HTLV antigens (Essex et al., 1983, Science, 220, 859-862).
Adult T-cell leukemia/lymphoma (ATL) occurs mainly in Southwestern Japan, the Caribbean basin and parts of Central and South America. In those regions, seroprevalence varies between 5% and 15% in the general population and reaches 30% in older age groups. In the United States, HTLV-I/II infections are mainly present in intravenous drug users (IVDU). In a recent American Red Cross survey (Williams et al., 1988, Science, 240, 643-646), antibodies to HTLV-I could be detected in 10 of 39,898 random blood donors in eight U.S. cities; this represents a seroprevalence rate of 0.025%. A similar study involving 3158 individuals from Northern Egypt led to the identification of two carriers (prevalence rate of 0.06%) (El-Farrash et al., 1988, Microbiol. Immunol., 32, 981-984). In these two studies, distinction between infection with HTLV-I and HTLV-II was not clearly established. These data indicate that there is a need for a reliable test to screen all blood samples destined for blood banks in order to avoid the inadvertent spread of the virus to blood product recipients.
There have been several attempts in the prior art to develop such tests. None has been successful in detecting all serum and plasma samples that are part of a well characterized commercial panel of HTLV infected fluids. Furthermore, none is able to detect HTLV infection at very low levels, thereby, ensuring safety of the blood supply and prompt and early treatment of HTLV infections.
Saxinger et al. (1984, Science, 225, 1473-1476) has reported the use of the HTLV-I particle as the immunoadsorbent in an enzyme immunoassay (EIA) for the detection of antibodies to the virus. In an improvement to this first generation test, specific HTLV antigens have been used instead of viral particles as the immunoadsorbent. For example, Samuel et al. (1984, Science, 225, 1094-1097) refers to antigens obtained by recombinant DNA technology. These detected each of 11 sera shown to contain antibodies to HTLV-I by a whole viral lysate-based EIA similar to the one developed by Saxinger et al. Slamon et al. (PCT/US85/01803) refer to assays using polypeptides and fragments thereof associated with immunogenic sites present on proteins of the pX region of HTLV-I and HTLV-II. The reported accuracy of these assays ranged between 77% and 87%. Fukui et al. (European Patent Application 87116787) refers to assays using polypeptides encoded by a fused gene comprising all or a part of the gag gene and all or a part of the env gene. They reported a sensitivity of 100% (57/57 ) with no false-positives.
Although these results look impressive, they are likely not repeatable with sera, like those most usual in the United States, which are characterized by much lower antibody titers than the Japanese sera used to validate the above assays.
Assays which use peptides derived from HTLV-I or HTLV-II proteins have also been reported. For example, Palker et al. (1989, J. Immunol., 142, 971-978) reports that antibodies in sera from 28 out of 36 patients (78%) reacted with a peptide spanning amino acids 190 to 209 of the HTLV-I envelope (peptide 4a; env 190-209).* Ten of 35 sera samples (29%) reacted with peptide 6 (env 296-312) and 6 out of 33 (18%) bound to peptide 7 (env 374-392). Palker et al. (1986, J. Immunol., 136, 2393-2397) also refers to a peptide (SP-71; gag 120-130) that reacted with 16 out of 18 HTLV-I seropositive samples. FNT *In this application, the amino acid sequence and numbering published by Seiki et al. (supra.) and by Shimotohno et al. (supra.) for the HTLV-I and HTLV-II gene products are used (for ease of reference only).
A peptide (SP-70; env 296-306) has been reported by Copeland et al. (1986, J. Immunol., 137, 2945-2951) to recognize antibodies from 4 out of 12 individuals seropositive for HTLV-I.
Wang et al. (U.S. Pat. No. 4,833,071) refers to three overlapping linear peptides spanning regions of the transmembrane protein (gp21) of HTLV-I. These peptides (env 381-400, env 377-400 and env 378-393), when used in a mixture, detected 102 out of 102 serum samples from patients with ATL and 5 out of 30 patients with AIDS/ARC. No immunoreactivity was found against sera from 12 normal subjects or from 12 patients with autoimmune diseases.
Reyes (PCT/W089/06543) refers to a non-glycosylated, 41-amino acid recombinant peptide antigen derived from the gp46 of HTLV-I. This recombinant peptide, env(163-203), is reported to have been used in a solid-phase assay for the determination of serum antibodies in six patients with HTLV-I infection.
Vahlne (PCT/W089/08664) refers to four synthetic peptides, A, B, C and H, spanning env(381-404), env(273-293), env(223-242) and env(176-199) of the envelope protein of HTLV-I. From the examples provided, it appears that only peptide A was useful in an ELISA for the detection of specific antibodies to HTLV-I present in the samples tested.
None of the above-described assays has demonstrated the high specificity (no false positives) and high sensitivity (detection of all positives, even when the sera contains very low levels of HTLV antibodies) necessary to ensure that HTLV infected blood products do not enter blood banks and that infected individuals seek prompt and early treatment. The peptides of this invention remedy these failures.